Interactions of ligands with cell surface receptor molecules are hallmarks of biological processes. Exemplary of such interactions are the binding of the ligand formed by a portion of the envelope protein (gp140) of the AIDS virus (HIV) with the CD4 receptor on T cells, the receptors of the major histocompatibility complex that interact with numerous ligands in the processes of self/non-self discrimination in the immunological system, and the interaction of the fibrinogen ligand with the GPIIb-IIIa cellular receptor on platelets. The fibrinogen:GPIlb-IIIa ligand-receptor pair are of particular interest in blood clotting, thrombus formation, and are utilized hereinafter as exemplary of ligands and receptors.
The art has long sought immunological methods to distinguish the bound and non-bound forms of ligands and receptors because that ability would allow for a determination of the state of the physiological mechanisms mediated by receptor:ligand complex formation. Until recently, efforts to make such determinations by immunological methods have been frustrated because biological samples can contain ligands and receptors in both bound (complexed) and free forms.
For instance, the vasculature of individuals undergoing a thrombotic event contains non-bound forms of fibrinogen and GPIIb-IIIa as well as platelets having a fibrinogen:GPIIb-IIIa complex on their surface. The fibrinogen:GPIIb-IIIa complex expresses antigenic determinants common to non-bound fibrinogen and non-bound GPIIb-IIIa, thus making it difficult to identify a thrombotic condition or thrombus location by existing immunological methods.
Recently, Frelinger et al., J. Biol. Chem., 263:12397-12402 (1988) reported identifying an antigenic determinant expressed by GPIIb-IIIa only when GPIIb-IIIa was bound to a ligand. That is, the antigenic determinant described by Frelinger et al. was expressed by the receptor of the receptor:ligand complex.
Others have reported the preparation of monoclonal antibodies that immunoreact with an antigenic determinant expressed upon ligand binding to artificial, non-receptor surfaces. Sofia et al., J. Colloid Interface Sci., 107:204-208 (1985) describe a particular monoclonal antibody, referred to as DSB.sup.2, induced by immunization with cross-linked fibrin fragment which binds to plastic-adsorbed fibrinogen and also to the so-called fibrinogen D fragment adsorbed on plastic or free in solution. Reportedly, the antibody does not bind to solution phase fibrinogen or the so-called fibrinogen E fragment when in solution.
Additionally, Nilsson et al., Molec. Immunol., 24:487-94 (1987), report the preparation of monoclonal antibodies that immunoreact with complement C3 fragments particle-bound to Zymosan A, but not with soluble C3 fragments. The nature of protein binding to Zymosan A involves covalent chemical bonds.
In another report Abrams et al., Blood (Suppl. 1), 70:355a (December 1987) briefly discuss a monoclonal antibody designated 9F9 that is said in the published abstract to bind to platelet-bound fibrinogen. That brief report, however, does not characterize tile specific binding properties of monoclonal 9F9.
Numerous monoclonal antibodies that immunoreact with solubilized fibrinogen have been reported. One such report is Lindon et al., Blood, 68:355-362 (Audi. 1988). That paper reported the use of commercially available anti-human fibrinogen monoclonal antibodies as well as affinity-purified polyclonal antibodies to the D and E fragments.